The present invention relates to the production of biologics such as viruses for vaccines, and more particularly to the harvesting of such biologics from eggs. Specifically, the present invention relates to apparatuses and methods for opening avian eggs and removing the desired biologics from within.
One method of producing biologics is to use fertilized avian eggs. The desired biologics are grown within the egg and must be harvested therefrom for further processing. Although a preferred embodiment of the present invention is directed to biologics such as viruses, the invention is believed to be applicable to other biologics that can be grown in eggs, such as proteins.
One method of producing vaccines, such as influenza vaccines, is to use fertilized avian (chicken) eggs. The eggs are injected with the viruses and, after a sufficient time of incubation to allow the virus to multiply, the eggs are opened to harvest the viruses.
Harvesting typically involves the collection of the allantoic fluid that is contained in the allantoic sac of a fertilized egg. It is preferable to harvest just the allantoic fluid and avoid contamination from the embryo containing yoke. The viruses are then separated from the fluid, purified, and inactivated to produce the final vaccine as is known in the art.
There are various methods for removing the allantoic fluid. One is to take advantage of the air sac within the top section of the egg shell. The top section, also referred to herein as the “egg cap”, can be cut to provide access to the allantoic fluid within. Various means can be utilized to remove the allantoic fluid for further processing.
As can be appreciated, it is desirable to be able to produce large quantities of vaccines as fast as possible. The present invention provides an advantageous apparatus and method for harvesting the allantoic fluid for producing vaccines.
Embryonated eggs have proven to be a useful medium for the isolation and identification of animal viruses, for titrating viruses, and for cultivation of viruses in the production of viral vaccines. The embryo, chorioallantoic membrane, yolk sac, allantoic sac, and amniotic sac may be inoculated in eggs at various developmental stages providing the scientist with large array of tissue types for specific purposes.
The apparatus and method of the present invention can be adapted for recovering a number of biologically active molecules from the components of embryonated avian eggs (e.g., allantoic fluid, embryo, chorioallantoic membrane, etc.) in addition to the influenza virus. Exemplarily biologically active molecules that may harvested from avian eggs components include viruses and immunoglobulins such as, but not limited to, flaviviruses (e.g., yellow fever virus); arboviruses (e.g., Sindbis virus, Murray Valley encephalitis virus, and Getah virus); orbiviruses (e.g., Bluetongue virus); aphtoviruses (e.g., type C foot-and-mouth-disease virus); alpharetrovirus (e.g., avian leukosis virus); gammaretrovirus (e.g., reticuloendotheliosis virus); rubulavirus (e.g., mumps virus and Newcastle disease virus); avian adenovirus (e.g., chicken embryo lethal orphan virus (CELO) and related quail bronchitis virus); infectious bronchitis the virus; and immunoglobulins from aves inoculated with a variety of infectious agents and/or antigens.
The production of viruses for influenza vaccine production is one preferred use of the present invention. The influenza viruses are some of the most ubiquitous viruses present in the world, affecting both humans and livestock. Influenza infections result in an economic burden, severe morbidity, and even death in the very young, the elderly and immunocompromised individuals. According to statistics from the World Health Organization, looking just at the U.S.A., there are 25-50 million cases of influenza resulting in approximately 150,000 hospitalizations and from 30,000-40,000 deaths per year. The world inter-pandemic influenza burden may be as high as 1 billion cases of influenza with 3-5 million cases of severe illness. Extrapolation of these statistics predicts from 300,000-500,000 annual deaths attributed to influenza worldwide.
Influenza viruses are spread from person to person, primarily through direct respiratory droplet transmission (e.g., when an infected person coughs or sneezes in close proximity to an uninfected person). Indirect transmission is also possible and usually results from tactical transfer (e.g., handshake) of contaminated secretion from an infected person to an uninfected person's nasal or conjunctival epithelium.
The typical incubation period for influenza is one to four days, with an average of two days. Adults can be infectious from the day before symptoms begin through approximately five days after illness onset. Children can be infectious for >10 days after the onset of symptoms, and young children also can shed virus before onset of illness. Severely immunocompromised persons can shed virus for weeks or even months after infection.
Uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e.g., fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis). Among children, otitis media, nausea, and vomiting also are commonly reported with influenza illness. Uncomplicated influenza illness typically resolves after three to seven days for the majority of persons, although cough and malaise can persist for >2 weeks. However, among certain persons, influenza can exacerbate underlying medical conditions (e.g., pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a coinfection with other viral or bacterial pathogens. Young children with influenza virus infection can have initial symptoms mimicking bacterial sepsis with high fevers, and febrile seizures have been reported in up to 20% of children hospitalized with influenza virus infection. Influenza virus infection also has been uncommonly associated with encephalopathy, transverse myelitis, myositis, myocarditis, pericarditis, and Reye syndrome.
Accordingly, improved methods and apparatuses for producing vaccines are desired.
Preferred embodiments of the present invention relate to methods and apparatuses for separating the components of avian eggs. Eggs suitable for use in the methods and apparatuses of the present invention can be obtained from a number of avian species including, but not limited to, domesticated chickens (gallus), turkeys, geese, ducks, quail, and the like. The present invention is primarily used to collect allantoic fluid from embryonated chicken eggs, however, the disclosed apparatuses and methods are useful for separating yolk and embryo from embryonated eggs as well. The embryogenesis of chick egg development is well characterized in the art. The reader is referred to standard texts in the field of chick development for additional details of the structures and development of chick embryos (e.g., R. Bellairs and M. Osmund, The Atlas of Chick Development, 2nd ed., Elsevier, New York N.Y., 2005).
The allantoic fluid from avian eggs, in particular chicken eggs, can be inoculated with live virus from the othomyxoviridae family. The inoculated virus replicates in the egg while the eggs are incubated from two to three days depending on the viral strain used for inoculation. The influenza virus is subsequently isolated and purified from the allantoic fluid collected from the inoculated eggs.
The othomyxoviridae family includes four genera: influenza A, influenza B, influenza C, and thogotovirus (sometimes called influenza D). Influenza A and B are responsible for most epidemic human disease. Influenza A also infects swine, horses, sea mammals, and birds, including, domesticated poultry and waterfowl. Human infection with influenza A usually results in more sever disease symptoms than those following infection with the other genera of influenza. Influenza A is also the most disposed to significant antigenic changes from season to season through antigen drifts and antigenic shift. Influenza B appears to only infect humans. Influenza C has been isolated from both swine and humans it is thought to cause only mild respiratory illness and not epidemics. Thogotoviruses are tick born viruses which are genetically and structurally related to the influenza A, B, and C viruses.
All othomyxoviridae viruses are enveloped viruses with a negative single stranded RNA (nsRNA) genome. In particular, influenza A and B viruses each contain eight segments of nsRNA enveloped in a glycolipid membrane derived from the host cell's plasma membrane. More particularly, the influenza A and B viral genome consists of segments PB2, PB1, PA, NP, M, NS, HA and NA) that encode at least 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1 and HA2), the matrix proteins (M1 and M2) and the non-structural proteins (NS1 and NS2) (Krug et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, N.Y., 1989, pp. 89-152).
The inner surface of the glycolipid membrane contains virus specific proteins while the exterior surface is studded with virus specific neuramidase (NA) and hemagglutinin (HA) proteins. HA was named for its ability to agglutinate erythrocytes (red blood cells) by attaching to N-acetylneuraminic (sialic) acid containing glycoprotein or glycolipid receptor sites on the surface of respiratory epithelial cells. HA is also responsible for facilitating penetration of the influenza virus particle into the cell's cytoplasm by mediating fusion of the virus particle membrane with the cell's membrane of the endosome encapsulating the virus particle with the consequence being the subsequent release of the viral nucleocapsids into the cell's cytoplasm. The nucleocapsid segments contain the viral genetic material destined for migration into the cell's nucleus. The acidic interior of the endosome encapsulating the virus particle causes the HA to slightly alter its structure and merge with the endosomal membrane until a hole is formed in the endosome. Major epidemics are associated with changes in the antigenic structure of HA and it is also the principal viral antigen against which infected hosts produce neutralizing antibodies. HA is the most important antigen in defining the serological specificity of the different influenza strains. This 75-80 kD protein contains numerous antigenic determinants, several of which are in regions that undergo sequence changes in different strains (strain-specific determinants) and others in regions which are common to many HA molecules (common to determinants).
NA is a hydrolytic enzyme that removes the terminal sialic acid from the cell's hemagglutinin receptors resulting in destruction of the receptor activity. The roles NA plays in influenza infection are not completely understood, however it is thought that NA may allow the virus particle to penetrate the mucin layer in respiratory tract that would otherwise bind virus particles and prevent them from contacting the surface of respiratory epithelial cells. NA may also be important in the fusion of the virus particle with the cell membrane prior to viral entry into the cell.
Influenza C virus is also enveloped with a nsRNA genome. The genome is composed of only seven RNA segments however and it has only a single multifunctional surface glycoprotein called hemagglutinin-esterase-fusion protein (HEF). As the names implies, the HEF protein has three functions a receptor-binding activity, a fusion activity, and a receptor-destroying activity.
Both influenza A and B viruses are further separated into groups on the basis of antigenic characteristics. Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: the hemagglutinin (H) and the neuraminidase (N). There are 16 different hemagglutinin subtypes and 9 different neuraminidase subtypes, all of which have been found among influenza A viruses in wild birds. Wild birds are the primary natural reservoir for all subtypes of influenza A viruses and are thought to be the source of influenza A viruses in all other animals. Most influenza viruses cause asymptomatic or mild infection in birds. Infection with certain avian influenza A viruses (for example, some strains of H5 and H7 viruses) can cause widespread disease and death among some species of wild and especially domestic birds such as chickens and turkeys. Only one subtype of HA and one of NA are recognized for influenza B viruses.
Influenza viruses can change in two different ways. One is called “antigenic drift.” These are small changes in the virus that happen continually over time. Antigenic drift produces new virus strains that may not be recognized by the body's immune system. This process works as follows: a person infected with a particular flu virus strain develops antibody against that virus. As newer virus strains appear, the antibodies against the older strains no longer recognize the “newer” virus, and reinfection can occur. This is one of the main reasons why people can get the flu more than one time. In most years, one or two of the three virus strains in the influenza vaccine are updated to keep up with the changes in the circulating flu viruses. So, people who want to be protected from flu need to get a flu shot every year.
The other type of change is called “antigenic shift.” Antigenic shift is an abrupt, major change in the influenza A viruses, resulting in new hemagglutinin and/or new hemagglutinin and neuraminidase proteins in influenza viruses that infect humans. Shift results in a new influenza A subtype. When shift happens, most people have little or no protection against the new virus. While influenza viruses are changing by antigenic drift all the time, antigenic shift happens only occasionally. Type A viruses undergo both kinds of changes; influenza type B viruses change only by the more gradual process of antigenic drift.
Pigs can be infected with both human and avian influenza viruses in addition to swine influenza viruses. Infected pigs get symptoms similar to humans, such as cough, fever, and runny nose. Because pigs are susceptible to avian, human and swine influenza viruses, they potentially may be infected with influenza viruses from different species (e.g., ducks and humans) at the same time. If this happens, it is possible for the genes of these viruses to mix and create a new virus. For example, if a pig were infected with a human influenza virus and an avian influenza virus at the same time, the viruses could mix (reassort) and produce a new virus that had most of the genes from the human virus, but a hemagglutinin and/or neuraminidase from the avian virus. The resulting new virus would likely be able to infect humans and spread from person to person, but it would have surface proteins (hemagglutinin and/or neuraminidase) not previously seen in influenza viruses that infect humans. This type of major change in the influenza A viruses is known as antigenic shift. Antigenic shift results when a new influenza A subtype to which most people have little or no immune protection infects humans. If this new virus causes illness in people and can be transmitted easily from person to person, an influenza pandemic can occur.
The term “avian” as used herein, is intended to include males and females of any avian species, but is primarily intended to encompass domestic poultry which is commercially raised for eggs, meat, or as pets. The term “avian” is particularly intended to encompass various avian species including, but not limited to, chickens, turkeys, ducks, geese, quail, pheasant, ostrich, and, emu, etc. Accordingly, the term “avian egg” refers to an embryonated egg laid by a female of one of the aforementioned avian species, and more preferably to an embryonated egg from a chicken.
As used herein, the term “membrane” refers to any layer of tissue within an egg that delimits an internal structure or area within the egg. Exemplary membranes within an egg include, but are not limited to, the outer shell membrane, inner shell membrane, the chorioallantoic membrane (CAM), vitelline membrane (VM), and amniotic membrane (amnion).
The present invention, which will now be described in detail below, provides novel methods and apparatuses for harvesting biologics from eggs.